Streptococcus pneumoniae is an important cause of otitis media, meningitis, bacteremia and pneumonia, and a leading cause of fatal infections in the elderly and persons with underlying medical conditions, such as pulmonary disease, liver disease, alcoholism, sickle cell, cerebrospinal fluid leaks, acquired immune deficiency syndrome (AIDS), and patients undergoing immunosuppressive therapy. It is also a leading cause of morbidity in young children. Pneumococcal infections cause approximately 40,000 deaths in the U.S. yearly. The most severe pneumococcal infections involve invasive meningitis and bacteremia infections, of which there are 3,000 and 50,000 cases annually, respectively.
Despite the use of antibiotics and vaccines, the prevalence of pneumococcal infections has declined little over the last twenty-five years; the case-fatality rate for bacteremia is reported to be 15-20% in the general population, 30-40% in the elderly, and 36% in inner-city African Americans. Less severe forms of pneumococcal disease are pneumonia, of which there are 500,000 cases annually in the U.S., and otitis media in children, of which there are an estimated 7,000,000 cases annually in the U.S. caused by pneumococcus. Strains of drug-resistant S. pneumoniae are becoming ever more common in the U.S. and worldwide. In some areas, as many as 30% of pneumococcal isolates are resistant to penicillin. The increase in antimicrobial resistant pneumococcus further emphasizes the need for preventing pneumococcal infections.
Pneumococcus asymptomatically colonizes the upper respiratory tract of normal individuals; disease often results from the spread of organisms from the nasopharynx to other tissues during opportunistic events. The incidence of carriage in humans varies with age and circumstances. Carrier rates in children are typically higher than those of adults. Studies have demonstrated that 38 to 60% of preschool children, 29 to 35% of grammar school children and 9 to 25% of junior high school children are carriers of pneumococcus. Among adults, the rate of carriage drops to 6% for those without children at home, and to 18 to 29% for those with children at home. It is not surprising that the higher rate of carriage in children than in adults parallels the incidence of pneumococcal disease in these populations.
An attractive goal for streptococcal vaccination is to reduce carriage in the vaccinated populations and subsequently reduce the incidence of pneumococcal disease. There is speculation that a reduction in pneumococcal carriage rates by vaccination could reduce the incidence of the disease in non-vaccinated individuals as well as vaccinated individuals. This "herd immunity" induced by vaccination against upper respiratory bacterial pathogens has been observed using the Haemophilus influenzae type b conjugate vaccines (Takala, A. K., et al., J. Infect. Dis. 1991; 164: 982-986; Takala, A. K., et al., Pediatr. Infect. Dis. J., 1993; 12: 593-599; Ward, J., et al., Vaccines, S. A. Plotkin and E. A. Mortimer, eds., 1994, pp. 337-386; Murphy, T. V., et al., J. Pediatr., 1993; 122; 517-523; and Mohle-Boetani, J. C., et al., Pediatr. Infect. Dis. J., 1993; 12: 589-593).
It is generally accepted that immunity to Streptococcus pneumoniae can be mediated by specific antibodies against the polysaccharide capsule of the pneumococcus. However, neonates and young children fail to make adequate immune response against most capsular polysaccharide antigens and can have repeated infections involving the same capsular serotype. One approach to immunizing infants against a number of encapsulated bacteria is to conjugate the capsular polysaccharide antigens to protein to make them immunogenic. This approach has been successful, for example, with Haemophilus influenzae b (see U.S. Pat. No. 4,496,538 to Gordon and U.S. Pat. No. 4,673,574 to Anderson).
However, there are over ninety known capsular serotypes of S. pneumoniae, of which twenty-three account for about 95% of the disease. For a pneumococcal polysaccharide-protein conjugate to be successful, the capsular types responsible for most pneumococcal infections would have to be made adequately immunogenic. This approach may be difficult, because the twenty-three polysaccharides included in the presently-available vaccine are not all adequately immunogenic, even in adults.
Protection mediated by anti-capsular polysaccharide antibody responses are restricted to the polysaccharide type. Different polysaccharide types differentially facilitate virulence in humans and other species. Pneumococcal vaccines have been developed by combining 23 different capsular polysaccharides that are the prevalent types of human pneumococcal disease. These 23 polysaccharide types have been used in a licensed pneumococcal vaccine since 1983 (D. S. Fedson and D. M. Musher, Vaccines, S. A. Plotkin and J. E. A. Montimer, eds., 1994, pp. 517-564). The licensed 23-valent polysaccharide vaccine has a reported efficacy of approximately 60% in preventing bacteremia by type pneumococci in healthy adults.
However, the efficacy of the vaccine has been controversial, and at times, the justification for the recommended use of the vaccine questioned. It has been speculated that the efficacy of this vaccine is negatively affected by having to combine 23 different antigens. Having a large number of antigens combined in a single formulation may negatively affect the antibody responses to individual types within this mixture because of antigenic competition. The efficacy is also affected by the fact that the 23 serotypes encompass all serological types associated with human infections and carriage.
An alternative approach for protecting children, and also the elderly, from pneumococcal infection would be to identify protein antigens that could elicit protective immune responses. Such proteins may serve as a vaccine by themselves, may be used in conjunction with successful polysaccharide-protein conjugates, or as carriers for polysaccharides.
McDaniel et al. (I), J. Exp. Med. 160:386-397, 1984, relates to the production of hybridoma antibodies that recognize cell surface polypeptide(s) on S. pneumoniae and protection of mice from infection with certain strains of encapsulated pneumococci by such antibodies.
This surface protein antigen has been termed "pneumococcal surface protein A", or "PspA" for short.
McDaniel et al. (II), Microbial Pathogenesis 1:519-531, 1986, relates to studies on the characterization of the PspA. Considerable diversity in the PspA molecule in different strains was found, as were differences in the epitopes recognized by different antibodies.
McDaniel et al. (III), J. Exp. Med. 165:381-394, 1987, relates to immunization of X-linked immunodeficient (XID) mice with non-encapsulated pneutnococci expressing PspA, but not isogenic pneumococci lacking PspA, protects mice from subsequent fatal infection with pneumococci.
McDaniel et al. (IV), Infect. Immun., 59:222-228, 1991, relates to immunization of mice with a recombinant full length fragment of PspA that is able to elicit protection against pneumococcal strains of capsular types 6A and 3.
Crain et al, Infect.Immun., 56:3293-3299, 1990, relates to a rabbit antiserum that detects PspA in 100% (n=95) of clinical and laboratory isolates of strains of S. pneumoniae. When reacted with seven monoclonal antibodies to PspA, fifty-seven S. pneumoniae isolates exhibited thirty-one different patterns of reactivity.
Above cited applications 08/529,055, filed Sep. 15, 1995, 08/470,626, filed Jun. 6, 1995, 08/467,852, filed Jun. 6, 1995, 08/469,434, filed Jun. 6, 1995, 08/468,718, filed Jun. 6, 1995, 08/247,491, filed May 23, 1994, 08/214,222, filed Mar. 17, 1994 and 08/214,164, filed Mar. 17, 1994, 08/246,636, filed May 20, 1994, and 08/319,795, filed Oct. 7, 1994, and U.S. Pat. No. 5,476,929, relate to vaccines comprising PspA and fragments thereof, methods for expressing DNA encoding PspA and fragments thereof, DNA encoding PspA and fragments thereof, the amino acid sequences of PspA and fragments thereof, compositions containing PspA and fragments thereof and methods of using such compositions.
PspA has been identified as a virulence factor and protective antigen. PspA is a cell surface molecule that is found on all clinical isolates, and the expression of PspA is required for the full virulence of pneumococci in mouse models (McDaniel et al., (III), J. Esp. Med. 165: 381-394, 1987). The biological function of PspA has not been well defined, although a preliminary report suggests that it may inhibit complement activation (Alonso DeVelasco, E., et al., Microbiological Rev. 1995; 59: 591-603).
The PspA protein type is independent of capsular type. It would seem that genetic mutation or exchange in the environment has allowed for the development of a large pool of strains which are highly diverse with respect to capsule, PspA, and possibly other molecules with variable structures. Variability of PspA's from different strains also is evident in their molecular weights, which range from 67 to 99 kD. The observed differences are stably inherited and are not the result of protein degradation.
Immunization with PspA in a lysate of a recombinant .lambda.gt11 clone, elicited protection against challenge with several S. pneumoniae strains representing different capsular and PspA types, as in McDaniel et al. (IV), Infect. Immun. 59:222-228, 1991. Although clones expressing PspA were constructed according to that paper, the product was insoluble and isolation from cell fragments following lysis was not possible.
Analysis of the nucleotide and amino acid sequences indicate that the PspA molecule comprises three major regions. The first 288 amino acids at the amino terminal end of the protein are predicted to have a strong alpha helical structure. The adjacent region of 90 amino acids (289 to 369 of Rx1 PspA) has a high density of proline residues; based on similar regions in other prokaryotic proteins, this region is believed to transverse the bacterial cell wall. The remaining 196 amino acids at the carboxyl-terminal end of the molecule (370 to 588 of Rx1 PspA) have a repeated amino acid sequence that has been demonstrated to bind to phosphocholine and lipoteichoic acids. Based on this structure, the PspA molecule is thought to associate with the inner membrane and lipoteichoic acids via the repeated region in the middle of the carboxyl-terminal end of the protein. The proline region in the middle of the protein is thought to transverse the cell wall placing the alpha helical region on the outer surface of the S. pneumoniae cells. This model is consistent with the demonstration that the alpha helical region, which extends from the surface of the cell, contains the protective epitopes (Yother, J. et al., J. Bacteriol. 1992; 174: 601-609; Yother, J. et al., J. Bacteriol. 1994; 176: 2976-2985; McDaniel, L. S. et al., Microbial Pathog. 1994; 17: 323-337; and Ralph, B. A., et al., Ann. N.Y. Acad. Sci. 1994; 730: 361-363).
Serological analysis of PspA using a panel of seven monoclonal antibodies, indicated that, like capsular polysaccharides, the PspA molecules are highly diverse among pneumococcal strains. Based on these analyses, over 30 PspA protein serotypes were defined, and assignments of individual strains into group using a classification system, i.e., families (or serotypes), were based upon reactivity with the panel of monoclonal antibodies. Moreover, SDS-PAGE analysis indicated that within a PspA serotype, further heterogeneity existed on the basis of the molecular size. This diversification further supports the assertion that PspA is a protective antigen in natural infections; the protective nature of anti-PspA responses has presumably applied selective pressure on pneumococcus to diversify this molecule. However, this diversification of the PspA molecule complicates the development of a PspA vaccine, and leads to the possibility that a PspA vaccine would have to contain many PspA strains, possibly making the vaccine impractical.
In addition to the published literature specifically referred to above, the inventors, in conjunction with co-workers, have published further details concerning PspA's, as follows:
1. Abstracts of 89th Annual Meeting of the American Society for Microbiology, p. 125, item D-257, May 1989;
2. Abstracts of 90th Annual Meeting of the American Society for Microbiology, p. 98, item D-106, May 1990;
3. Abstracts of 3rd International ASM Conference on Streptococcal Genetics, p. 11, item 12, June 1990;
4. Talkington et al, Infect. Immun. 59:1285-1289, 1991;
5. Yother et al (I), J. Bacteriol. 174:601-609, 1992; and
6. Yother et al (II), J. Bacteriol. 174:610-618, 1992. 7. McDaniel et al (V), Microbiol. Pathogenesis, 13:261-268, 1994.
Alternative vaccination strategies are desirable as such provide alternative routes to administration or alternative routes to responses. It would be advantageous to provide an immunological composition or vaccination regimen which elicits protection against various diversified pneumococcal strains, without having to combine a large number of possibly competitive antigens within the same formulation.